Summary

In the developing retina, the production of ganglion cells is dependent on
the proneural proteins NGN2 and ATH5, whose activities define stages along the
pathway converting progenitors into newborn neurons. Crossregulatory
interactions between NGN2, ATH5 and HES1 maintain the uncommitted status of
ATH5-expressing cells during progenitor patterning, and later on regulate the
transition from competence to cell fate commitment. Prior to exiting the cell
cycle, a subset of progenitors is selected from the pool of ATH5-expressing
cells to go through a crucial step in the acquisition of a definitive retinal
ganglion cell fate. The selected cells are those in which the upregulation of
NGN2, the downregulation of HES1 and the autostimulation of ATH5 are
coordinated with the progression of progenitors through the last cell cycle.
This coordinated pattern initiates the transcription of ganglion cell-specific
traits and determines the size of the ganglion cell population.

Introduction

The early development of the vertebrate central nervous system crucially
depends on the timely generation of specific classes of neurons at distinct
positions and in appropriate numbers. A large set of different regulatory
proteins are known to be implicated in neurogenesis, and considerable progress
has been made in piecing together the pathways specifying both the generic
neuronal traits and the subtype identity traits
(Anderson, 2001;
Bertrand et al., 2002;
Jessell, 2000). However, the
underlying transcriptional networks through which neuronal identity, number
and position are coordinately regulated remain poorly defined.

The retina is one region of the central nervous system in which the
conversion of progenitor cells into particular classes of neural cells is
quite well understood (Cepko,
1999; Harris and Holt,
1990; Reh and Levine,
1998). Retina ontogenesis is geared to generate glia and six
classes of retinal neurons from an undifferentiated neuroepithelium, according
to a program that controls proliferation, specification, exit from the cell
cycle and differentiation. Cell differentiation initiates in the inner layer
of the central optic cup and progresses radially to the peripheral edge of the
retina. A characteristic feature of vertebrate retinogenesis is that the
different retinal cell types are generated in a fixed sequence. Retinal
ganglion cells (RGC) differentiate first, followed in overlapping phases by
amacrine cells, horizontal cells, cone photoreceptors, rod photoreceptors,
bipolar cells and, finally, Müller glial cells.

The generic programs of neuronal differentiation are regulated in
vertebrates as in Drosophila
(Anderson and Jan, 1997) by
members of the basic helix-loop-helix (bHLH) class of transcription factors.
The achaete-scute homologue ASH1 and the three neurogenins
(NGN1-NGN3) are among the earliest bHLH genes expressed in the developing
nervous system and they are thought to act as proneural genes. A spatial
complementarity between the expression of ASH1 and of the neurogenins appears
to be the rule in most proliferating neuroepithelia and these factors have a
role in the ontogeny of distinct classes of progenitors
(Bertrand et al., 2002). In the
developing retina, most of the broadly expressed neurogenic bHLH proteins are
likewise implicated in the generation of distinct classes of neurons
(Inoue et al., 2002;
Vetter and Brown, 2001), but
it is unresolved whether these factors act individually or combine to promote
particular neuronal phenotypes. Likewise, the molecular mechanisms that
control the timing of their expression and/or function are poorly defined.

Lineage tracing studies have led to the hypothesis that retinal progenitors
pass through a series of different competence states during which they
sequentially produce different types of neural cells
(Livesey and Cepko, 2001).
This model suggests that progenitors may be limited to producing certain types
of neurons at a given time in the course of retinogenesis. Here, we attempt to
define at the molecular level the time frame and cellular context in which
progenitors yield RGCs in the developing chick retina. Specifically, we
identify several of the stages along the pathway leading to the conversion of
progenitors into newborn RGCs. We show how the interplay between ATH5 and
other bHLH proteins controls the transitions between stages and coordinates
RGC specification with the patterning of progenitor cells. We highlight a
program that operates during the two main phases of ATH5 expression,
coordinating the selection of RGC precursors and the induction of RGC-specific
traits with cell cycle exit. The first phase involves crossregulatory
interactions between ATH5, NGN2, ASH1 and HES1 proteins that allow the
expansion of pools of progenitors, contribute to their progressive
intermingling and maintain ATH5 expression below the level required for
inducing RGC differentiation. The second phase initiates when RGC progenitors
are dispersed throughout the retina. The coordinated upregulation of NGN2 and
downregulation of HES1 contribute to the progression of progenitors through
the last cell cycle and create a suitable environment for efficient ATH5
autostimulation. The ATH5 protein upregulates its own expression and initiates
the transcription of RGC-specific traits. Cells committed to the RGC fate then
exit the cell cycle and express post-mitotic neuronal markers. In sum, we show
how a subset of progenitors is selected from the pool of ATH5-expressing cells
to enter the specification pathway at the proper time for RGC genesis.

Materials and methods

Reporter plasmids for the ATH5 and β3 promoters

A fragment of the ATH5 gene, 912 bp in length and bounded by XbaI
and BstXI restriction sites (GenBank AJ630209) was subcloned in the
proper orientation at appropriate sites in the vectors p00-CAT, p00-lacZ and
p00-GFP to yield, respectively, p00-ATH5-CAT, p00-ATH5-lacZ and p00-ATH5-GFP.
The similarly constructed p00-β3-lacZ plasmid bears the 143 bp promoter
of the gene encoding the neuronal acetylcholine receptor β3 subunit and
has been described previously (GenBank X83740).

Eukaryotic expression plasmids for ATH5, NGN2 and HES1

The pEMSV plasmid (Matter-Sadzinski et
al., 2001), which puts a cloned sequence under the transcriptional
control of the mouse sarcoma virus long terminal repeat, was used throughout
to express the ATH5, NGN2 and HES1 cDNAs in co-transfection and
electroporation experiments.

In situ hybridization

35S-labelled antisense riboprobes were synthesized and in situ
hybridization on tissue sections were performed as described by
Matter-Sadzinski et al. (Matter-Sadzinski
et al., 2001). To correlate the expression level of a particular
gene with ATH5 or β3 promoter activity, transfected retinal cells were
stained for β-galactosidase and processed for in situ hybridization as
described by Matter-Sadzinski et al.
(Matter-Sadzinski et al.,
2001). For quantification (Fig.
1O), silver grains were counted in 50 radial sectors (∼1300μ
m2 each) corresponding to a visual angle of ∼3°.

[3H]-thymidine and BrdU labelling

To label the S phase, transfected cells were incubated in medium containing
5 μCi/ml [3H]-thymidine for 3 hours (stage 22-23) or 1 hour
(stage 29-30) at the end of the 24 hours expression period. They were stained
for β-galactosidase and processed for autoradiography
(Matter-Sadzinski et al.,
2001). Neuroretinas were dissected and incubated for 45 minutes in
medium containing 100 μM BrdU and chased for 15 minutes. The explants were
fixed, embedded in paraffin wax, sectioned and processed for in situ
hybridization and for immunodetection of BrdU
(Roztocil et al., 1997).

Cell cultures, transfection, CAT and β-galactosidase assays

Chick embryos were staged according to Hamburger and Hamilton (Hamburger
and Hamilton, 1951). Neuroretina from stage 22-23 to stage 38 embryos were
dissected and dissociated into single cells that were transfected with CAT,
lacZ or GFP reporter plasmids. All transfections were carried out using the
lipofectin reagent (InVitrogen), as described by Matter-Sadzinski et al.
(Matter-Sadzinski et al.,
1992). In all instances, the ratio of DNA to lipofectin was 1:4.
In transfection experiments using a single construct, we transfected 1 μg
of reporter plasmid per 106 cells. In co-transfection experiments
using two or three constructs, 1 μg of reporter plasmid was mixed,
respectively, with 0.5 μg or 2×0.5 μg expression vectors per
106 cells. Negative controls consisted of 1.0 μg reporter
plasmid and 1.0 μg empty expression vector per 106 cells.
Quantification of the chloramphenicol acetyl transferase (CAT) activity
obtained with pATH5-CAT and identification of β-galactosidase-positive
cells (lacZ) were as described by Matter et al.
(Matter et al., 1995) and
Matter-Sadzinski et al. (Matter-Sadzinski
et al., 2001).

Electroporation of genetic material in the retina

Retinas were prepared from embryonic eyes collected at stages 22-23.
Electroporations were as described by Matter-Sadzinski et al.
(Matter-Sadzinski et al.,
2001). Briefly, whole retinas or dissected peripheral and central
sectors were immersed at room temperature in phosphate-buffered saline
containing a reporter plasmid and/or expression vectors (100 μg/ml of each
construct). Electroporation consisted of five 50 V/cm pulses of 50 mseconds
duration spaced 1 second apart. The electroporated tissues were cultured as
floating explants for 24 hours at 37°C. GFP- andβ
-galactosidase-positive cells were revealed or tissues were frozen in
liquid nitrogen prior to RNA extraction.

Single-cell collection and RT-PCR

Cells transfected with the ATH5-promoter/GFP reporter plasmid were plated
into poly-DL-ornithine-coated plastic petri dishes (30 mm in diameter).
Twenty-four or 48 hours after transfection, individual GFP-positive cells were
collected by aspiration with a glass micropipette mounted on a
micromanipulator. Single-cell RT-PCRs were performed according to Brady and
Iscove (Brady and Iscove,
1993). Each cell was collected in 10 μl of a buffer containing
50 mM Tris HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT
supplemented with 10 U of RNAsin (Promega), and immediately frozen in liquid
nitrogen. Single cells were thawed on ice, vortexed for 10 seconds, spun down,
heated to 65°C for 1 minute, vortexed again and then incubated on ice for
1 minute. For reverse transcription, dNTPs and Nonidet P-40 were added to
final concentrations of 0.5 mM each dNTP, 0.5% NP-40, supplemented with 50 ng
random DNA hexamers and 10 U RNAsin. 5 U of DNAse 1 (Gibco) was added and the
mix was incubated at 37°C for 30 minutes to destroy genomic DNA. DNAse 1
was inactivated by 10 minutes incubation at 65°C. The mix was aliquoted in
two fractions, both of which were again treated with 10 U of RNAsin and one of
which received 200 U of Superscript II reverse transcriptase (Gibco). Both
samples were incubated at 25°C for 10 minutes, at 37°C for 1 hour and
at 68°C for 10 minutes. The whole reaction mixes were then used as
templates in a first PCR performed with ExTaq polymerase (TaKaRa) and the
complete set of external primers (0.2 mM final concentration of each primer)
designed for amplifying the genes of interest (see Table S1 in the
supplementary material). Initial denaturation at 94°C for 3 minutes was
followed by 35 cycles consisting of denaturation at 94°C for 40 seconds,
annealing at 56°C for 40 seconds, elongation at 72°C for 1 minute, and
a final elongation at 72°C for 3 minutes. The second PCR was performed
separately for each gene of interest using the internal primers specific for
this gene (4 mM final concentration of each primer) and 0.1 volume of the
first PCR as template. PCR conditions were as described above. When using the
ATH5 internal primers (see Table S1 in the supplementary material), PCRs were
performed with templates originating from both the actual and the mock reverse
transcriptions. Only those cells that yielded no amplification of the negative
control sample were selected for expression of the other genes of
interest.

Several bHLH factors pattern the early retinal neuroepithelium. (A) At
stage 14, HES1 transcripts accumulate in discrete domains in the eyecup (ec)
and neural tube (nt). There is no detectable accumulation of HES1 transcripts
in the central region (red bracket) of the presumptive retina. (B,C) The first
ATH5- and NGN2-expressing cells are detected in the central retina (nr) at
stage 15. (D) At stage 16, ASH1 transcripts are not detected in retina. (E) At
stage 17, a robust accumulation of HES1 transcripts is taking place throughout
the peripheral retina (J). In the central retina, a few cells located on the
vitreous side express HES1 at a high level (arrowheads in E,I). (F,K) Most
cells in the central retina express ATH5 and those expressing ATH5 strongly
are mostly located on the vitreous side. (H) At stage 18, the sparse cells
expressing Neuro M are scattered across the central retina (arrowhead in M).
There are no cells expressing ATH5 or Neuro M in the HES1 domain (F,H,L,N).
(O) Quantification of in situ hybridization. Adjacent retinal sections were
hybridized with the indicated bHLH riboprobes at stage 18. The ATH5, Neuro M
and NGN2 domains coincide in the central retina and they abut on the
peripheral HES1 domain. ASH1 is detected in an annular sector (G, brackets) at
the interface between the HES1 and ATH5 domains. l, lens. Scale bar: 140 μm
in A; 80 μm in B,C; 100 μm in D-H.

Results

The early retinal neuroepithelium is patterned by distinct bHLH
expression domains

The spatial analysis of bHLH gene expression in early retina reveals a
remarkable diversity of progenitor populations
(Fig. 1). At stage 14 (E2), the
structural continuum between the retinal neurepithelium and the neural tube is
still in place, and HES1 transcripts accumulate in discrete territories of
both structures (Fig. 1A). In
the inner layer of the eyecup, the HES1 expression domain includes the
periphery of the presumptive neuroretina and there is no marked accumulation
of HES1 transcripts in the central region, where the first ATH5- and
NGN2-expressing cells appear at stage 15-16
(Fig. 1B,C). During the next 12
hours – between stages 15 and 18 – a robust accumulation of HES1
transcripts is taking place in a peripheral domain expanding to the anterior
edge of the retina (Fig. 1E,J),
whereas the central region expresses HES1 at a low level, except for a few
cells located on the vitreous side (Fig.
1E,I). As revealed by cell counting on serial sections of stage 18
retinas, ∼88% of cells (370±20 cells/section) in the central domain
express ATH5 and these cells represent ∼26% of the total retinal cell
population (Fig. 1K,
Fig. 2A)
(Skowronska-Krawczyk et al.,
2004). About 1.5% of cells express ATH5 at a high level and they,
as well as the sparse cells (∼1%) expressing the post-mitotic bHLH Neuro M
(Fig. 1H,M), are evenly
distributed throughout the central retina. The Neuro M-expressing cells
comprise the first set of newborn neurons, and their homogeneous distribution
(Fig. 1H) indicates that
neurogenesis is initiated at the same rate throughout the central domain. The
ATH5 and NGN2 domains precisely coincide at this stage, and they border the
peripheral HES1 expression domain (Fig.
1O). Expression of ASH1 is undetectable in retina until stage 18,
at which point an annular ASH1 region surrounds the central domain
(Matter-Sadzinski et al.,
2001) and overlaps the posterior edge of the HES1 domain
(Fig. 1D,G,O). Very few cells
expressing ATH5 or NGN2, and no Neuro M positive cells were found in the ASH1
or HES1 expression domains (Fig.
1L,N), confirming that the early retinal neuroepithelium is
patterned in discrete progenitor domains between stages 14 and 18.

The concentric expression pattern of the bHLH genes is maintained until
stage 26 (E5) (Fig. 2).
However, from stage 23 onwards, dynamic changes are taking place. Between
stages 18 and 26, the retina diameter increases about threefold and expansion
of the ATH5 and NGN2 expression domains parallels the growth of the whole
retina (Fig. 2A-E). HES1
expression, on the whole, remains complementary to that of ATH5, with
transcript levels maintained very high at the periphery and low in the centre
(Fig. 2C). At stage 23, the
central retina still contains isolated cells expressing HES1 at a high level
(Fig. 2L). Contrasting with the
mutually exclusive domains established at earlier stages, the anterior margin
of the ATH5 domain now overlaps the posterior HES1 region, where the levels of
HES1 transcripts are decreasing (Fig.
2H,I). Moreover, from stage 23 onwards, the NGN2 domain expands
more peripherally than that of ATH5, suggesting that NGN2 expression is less
sensitive than ATH5 to inhibition by HES1
(Fig. 2J,K), and thus precedes
the onset of ATH5 expression as both domains expand to the periphery.
Expansion of the NGN2 and ATH5 domains is paralleled by changes in the
expression pattern of ASH1. At stages 23-26, an annular ASH1 expression region
is still surrounding the ATH5 domain but ASH1- and ATH5/NGN2-expressing cells
are intermingled in the centre (Fig.
2E,F). At stages 28-30 (E6), distinct progenitor domains are no
longer detected and ATH5-expressing cells are distributed throughout the
retina, except at the ciliary margin (Fig.
2G). Expansion of the ATH5 domain to the periphery is paralleled
by a strong increase in accumulation of ATH5 mRNA
(Fig. 2G) and coincides with
the downregulation of HES1 in central and peripheral retina
(Fig. 2M). Thus, dynamic
changes in the expression profile of progenitor cells at domain boundaries and
in the central retina lead to a blending of different precursor sets, to a
progressive blurring of borders and to the merging of formerly discrete
domains.

Growth of the retina is accompanied by changes in the patterning of
progenitor cells. (A,B,E) Between stages 18 and 26, the ATH5 domain expands in
register with the threefold increase in retina diameter. (C) HES1 transcripts
are abundant at the periphery and sparse in the central region. Scattered
cells expressing HES1 at a high level are detected in the central retina
(arrowheads in L). The HES1 and ATH5 expression domains are complementary
(B,C), but the anterior margin of the ATH5 domain overlaps the posterior HES1
region, where HES1 transcript levels are decreasing (brackets in H,I). ATH5
and NGN2 transcripts accumulate in the posterior retina (B,D). The NGN2 domain
extends beyond that of ATH5 (brackets in J,K). At stage 26, ASH1- and
ATH5-expressing cells are interspersed in the posterior retina. ASH1 extends
beyond ATH5 (arrowheads in E,F). At stage 30, ATH5 transcripts are distributed
throughout the whole retina (G), except at the ciliary margin (arrows). ATH5
transcripts are not evenly distributed across the retina. They are abundant on
the ventricular side of the proliferative zone (pz) (inset in G). At stage 30,
HES1 expression is downregulated both in the peripheral and in the central
retina (M). Sections in L and M were counterstained with Toluidine Blue. Scale
bar: 380 μm in A-G; 40 μm in L; 60 μm in M; 240 μm in H,I; 150μ
m in J,K.

Activity of the electroporated ATH5 promoter in stage 22-23 retina. (A)
When controlled by the ubiquitous CMV promoter, GFP and lac reporters
are both expressed in the electroporated peripheral retina. (B-F) The
ATH5-promoter/lac and the CMV-promoter/GFP reporter plasmids were
electroporated alone (B,C), in combination with a NGN2 expression vector (D,E)
or in combination with NGN2 and HES1 expression vectors (F). (B) GFP-positive
cells are distributed throughout the peripheral (p) and central (c) retina,
whereas lac+ cells are confined to the central region
(arrowhead). (C) lac+ cells (arrowheads) are sparse in the
central retina. (D) Overexpression of NGN2 increases the proportion of
lac+ cells in the central, but not in the peripheral
retina (arrowhead in E). (F) No lac+ cells were detected
when both NGN2 and HES1 were overexpressed in the central retina. Data
presented in each panel are representative of at least five independent
experiments. l, lens. Scale bar: 170 μm in A; 120 μm in B; 30 μm in
C; 40 μm in D,F; 80 μm in E.

The spatiotemporal expression of ATH5 is regulated at the promoter
level

The cis-regulatory region of the chick ATH5 gene extending 775 bp
upstream of the translation initiation codon (GenBank AJ630209) contains
important regulatory elements and this region drives reporter activity in
those cells where in vivo ATH5 mRNA accumulation takes place during
retina development (Matter-Sadzinski et
al., 2001) (J.H., L.M.-S., D. Skowronska-Krawczyk, J.-M.M. and
M.B., unpublished). To determine whether the isolated promoter reproduces the
spatiotemporal pattern of gene regulation at early developmental stages, a mix
of an ATH5-promoter/lacZ-reporter plasmid with a CMV-promoter/GFP
reporter construct was electroporated in retinas at stage 22-23. After 24
hours, GFP-positive cells were distributed throughout the central and
peripheral retina, whereas lac+ cells were confined to the
central domain, as expected at this early stage
(Fig. 3B,C). The rather low
density of lac+ cells in this experiment is due to the
relative insensitivity of the X-gal assay, which, as we show below, only
detects promoter activity in cells that express ATH5 at a high rate.

Single-cell transcription analysis reveals the stages of a
progression along the RGC specification and differentiation pathway

The isolated ATH5 promoter region provides a unique means of identifying
ATH5-expressing progenitor cells, some of which will become committed to the
RGC lineage. Whereas in situ hybridization suffices to colocalize promoter
activity and expression of a single gene in individual cells
(Matter-Sadzinski et al.,
2001), the single-cell RT-PCR approach is necessary for detecting
the co-expression of multiple genes. To monitor the dynamics of bHLH
expression in individual cells during the period of RGC specification and
differentiation, acutely dissociated cells from stage 22-23 (E3.5) and 26 (E5)
retinas were transfected with the ATH5-promoter/GFP-reporter plasmid singly or
in combination with a NGN2 expression vector, plated into tissue culture
dishes and cultured for 24 or 48 hours. The time of cell collection thus
approximately corresponded to E4.5, E5.5 and E6 in vivo. One-hundred and sixty
GFP-positive cells were collected and single-cell RT-PCRs were performed to
produce collections of cDNA fragments representing the mRNA of single
ATH5-expressing cells. To examine the combinations in which the selected genes
were expressed, cells from the five groups generated by the experiment
(Fig. 4B) were independently
tested by second rounds of PCR, using appropriate (see Table S1 in the
supplementary material) sets of primers
(Fig. 4A,
Fig. 5A,B). All GFP-positive
cells expressed ATH5, as expected (Fig.
4A), but there was a high degree of heterogeneity and striking
temporal changes in the expression profiles of the unselected genes HES1,
Delta 1, Neuro M, β3nAChR and BRN3C. At early stages (E4.5), we did not
find any cells co-expressing ASH1 and ATH5 (0/20), confirming that these two
proneural genes are initially expressed in separate sets of early progenitors.
At later stages (E5.5-E6), three out of 28 cells expressed both genes (e.g.
cell 149), two of them expressing Neuro M as well (data not shown). The large
majority (∼80%) of cells transfected at stage 22-23 and collected 24 hours
later were expressing HES1, evidence that most ATH5-expressing cells are
proliferating progenitors. This proportion decreased to ∼5% when stage
22-23 cells were kept for 48 hours in culture or when cells were transfected
at stage 26 (Fig. 4C).
Conversely, 10% of cells transfected at stage 22-23 were found to express ATH5
and Neuro M after 24 hours in culture. This proportion increased to ∼60%
when stage 22-23 cells were cultured for 48 hours (e.g. cell 128), or when
stage 26 cells were cultured for 24 hours (e.g. cell 54). The expansion of the
population expressing Neuro M is paralleled by a proportional decrease in the
number of HES1-expressing cells as RGC precursors exit the mitotic cycle, a
process culminating at E6. Because no cells co-expressing HES1 and Neuro M
were found in the collection (0/60), whereas numerous single cells expressed
neither HES1 nor Neuro M (22/60), these two proteins must be expressed at
distinct stages separated by a lag. Interestingly, overexpression of NGN2 at
stage 22-23 leads, after 24 hours, to a drastic decrease in the proportion of
HES1-positive cells, but does not induce the precocious generation of Neuro
M-positive cells (Fig. 4C),
resulting instead in the accumulation of cells that express neither HES1 nor
Neuro M. The proportion of Neuro M-positive cells increased to ∼60% when
stage 22-23 cells overexpressing NGN2 were cultured for 48 hours. Some cells
co-expressed Delta 1 and HES1 (e.g. cell 114) and Delta 1 expression was seen
both in the presence of Neuro M (e.g. cell 126) and in cells that expressed
neither Neuro M nor HES1 (e.g. cell 116), indicating that the Notch ligand is
expressed soon after the downregulation of HES1 but prior to the onset of
Neuro M expression, a result that is consistent with previous in situ
hybridization studies (Henrique et al.,
1997; Roztocil et al.,
1997). The small proportion of Delta 1-positive cells (4/39;
Fig. 4A) suggests that this
gene is expressed very transiently in differentiating RGCs.

Transcriptional analysis of ATH5-expressing single cells. Stage 22-23
(E3.5) retinal cells were transfected with an ATH5-promoter/GFP-reporter
plasmid either singly or in combination with a vector expressing NGN2. They
were cultured for either 24 (E4.5) or 48 hours (E5.5). Stage 26 (E5) retinal
cells were transfected with the ATH5-promoter/GFP-reporter plasmid and
cultured for 24 hours (E6). Individual GFP-positive cells were collected and
processed for single-cell RT-PCR using the primers listed in Table S1 (see
supplementary material). (A) Representative transcriptional profiles obtained
with a set of 39 cells from the five groups generated by the experiment, as
identified by the colour code in B. RT-PCRs of total RNA isolated from E8
retina (NRE8) were used as positive controls for each set of primers. (C)
Ratios of HES1-, Neuro M-, Delta 1- and ASH1-positive cells to the total
number of cells tested for expression of these genes.

Co-expression of ATH5, Neuro M and of the RGC-specific genes β3nAChR
and BRN3C was mostly detected in stage 26 cells cultured for 24 hours,
indicating that these post-mitotic cells are newborn RGCs
(Fig. 5A,B). The presence ofβ
3 transcripts in cells that do not express Neuro M (e.g. cells 49, 136)
is consistent with a previous report demonstrating that the β3 promoter
is activated in cells that are still proliferating
(Matter et al., 1995). This is
supported by the following additional findings: when retinal cells were
transfected at stage 24 with a β3-promoter/lacZ reporter
plasmid, low levels of HES1 transcripts were detected in ∼10% of
lac+ cells (Fig.
5C, part a). The β3 promoter generally was more active in
cells that did not express Neuro M and its activity decreased in newborn RGCs
(Fig. 5C, parts b and c), in
keeping with previous studies showing that the β3 promoter peaks between
E5 and E6 – i.e. about 12 hours before the maximum level of ATH5
expression is reached (Matter et al.,
1995; Matter-Sadzinski et al.,
2001). No ATH5-positive single cell expressed BRN3C (0/20) when
stage 22-23 cells were cultured for 24 hours (data not shown). BRN3C
expression was seen only when stage 22-23 cells were cultured for 48 hours, or
when stage 26 cells were cultured for 24 hours and all BRN3C-positive cells
expressed Neuro M (e.g. cells 39 and 132). The small proportion of Neuro
M-positive cells expressing BRN3C (3/17;
Fig. 5 and data not shown)
suggests that this gene is turned on when cells have exited the cell cycle and
relatively late after the onset of Neuro M expression. This result is
congruent with the reported presence of BRN3C in migrating and differentiated
RGCs (Liu et al., 2000).
Overall, the particular combinations of genes and their temporal expression
profiles, as revealed by single-cell PCR are in good agreement with the
genetic sequence of development in the ciliary margin of Xenopus
retina (Perron et al., 1998).
As judged by their expression profiles, ATH5-expressing cells fall into three
main groups. First, there are HES1-expressing proliferating cells. Second,
there are cells that do not express HES1 but express β3 and/or Delta 1.
These are presumably passing through the last cell cycle. Third, there are
post-mitotic cells expressing Neuro M. These cells correspond to newborn RGCs
and some of them express BRN3C. Cells showing mixed status most probably were
captured at the juncture between two phases (e.g. cells co-expressing HES1 and
Delta 1) or in a transient state prior to acquiring a definite progenitor
status (e.g. cells co-expressing ATH5 and ASH1).

Co-expression of RGC-specific genes and bHLH transcription factors in
newborn RGCs and RGC precursors. Cells were transfected with an
ATH5-promoter/GFP-reporter plasmid at stage 26 (E5) and cultured for 24 hours.
(A, right) Transcriptional profile of a newborn RGC. This neuron-like
GFP-positive cell (left) expresses ATH5, Neuro M, β3 and BRN3C, but not
ASH1. (B) ATH5-expressing cells do not always co-express Neuro M, β3 and
BRN3C. (C) Colocalization of β3 promoter activity and HES1 or Neuro M
expression. Cells were transfected with aβ
3-promoter/lacZ-reporter plasmid at stage 24. After 24 hours in
culture, lacZ-expression was revealed and cells were processed for in
situ hybridization with (a) HES1- or (b,c) Neuro M-specific riboprobes.
Arrowhead in a indicates a double-labelled cell.

Transcription of the ATH5 gene is regulated in several distinct
phases

We then asked how ATH5 expression is regulated along the course of RGC
specification. Promoter activity and accumulation of mRNA follow the same
kinetics, indicating that the differential expression of ATH5 during retina
development is regulated at the transcriptional level
(Matter-Sadzinski et al.,
2001) (Fig. 6A).
The similar proportions of retinal cells expressing ATH5 at stages 18
(∼26%) and 29-30 (∼33%)
(Skowronska-Krawczyk et al.,
2004) suggest that the expression of ATH5 essentially reflects
changes in the transcription rate within a roughly constant cell fraction,
thus validating the analysis of promoter activity in the course of early
retina development. The analysis revealed that transcription of the ATH5 gene
is regulated in three sequential phases
(Fig. 6). The first phase
extends from E2 to E5 and is marked by low promoter activity. Because ATH5 and
NGN2 contribute to the regulation of the ATH5 promoter
(Fig. 3)
(Matter-Sadzinski et al.,
2001), we asked whether overexpression of these proteins might
modify promoter activity at early developmental stages. Retinal cells were
co-transfected at stages 22-23 or 23-24 with an ATH5-promoter/CAT reporter
plasmid and an expression vector encoding either ATH5 or NGN2. Whereas
overexpression of NGN2 resulted in a strong increase in promoter activity,
ATH5 had no significant effect (Fig.
6A). In a complementary experiment, the number of
lac+ cells markedly increased when the NGN2 expression
vector was mixed with the ATH5-promoter/lacZ reporter plasmid prior
to electroporation in stage 22-23 retina
(Fig. 3D). This effect was not
detected when an ATH5 expression vector was mixed to the ATH5
promoter/lacZ reporter plasmid. Activation of the ATH5 gene
by NGN2 was independently demonstrated by electroporating the NGN2 expression
vector into stage 22-23 central retinas, which led to a strong accumulation of
endogenous ATH5 mRNA (Fig. 7B),
whereas that transcript was barely detectable in the control. These results
suggest that ATH5 expression is weak in early retina because NGN2 is expressed
at an insufficient rate and because ATH5 is inefficient at activating its own
promoter at early stages. Significant changes in the behaviour of the ATH5
promoter were detected at stage 25-26. The transfected promoter, like the
endogenous one, is still poorly active but ATH5 overexpression upregulates it
to the level it would normally reach on E6, indicating that the cellular
context is now permissive for ATH5 autostimulation
(Fig. 6A). This transition
coincides with the suppression of HES1 expression in the majority of
ATH5-expressing cells (Fig. 4).
Promoter activity rapidly increases after stage 26 and can be further enhanced
by overexpression of either ATH5 or NGN2, the effect of ATH5 being more
pronounced than that of NGN2 (Fig.
6A). At stage 29-30, ATH5 transcripts accumulate in ∼33% of
retinal cells and transfection by the CAT reporter reveals a robust increase
in promoter activity. The lacZ reporter, however, labels only∼
10% of the transfected cells (Fig.
6B,D). This discrepancy comes from a difference in the sensitivity
of the techniques. Compared with the CAT assay, which monitors promoter
activity in virtually all ATH5-expressing cells, X-gal staining only detects
expression of the reporter gene above a threshold level, which was not reached
at this stage in about two-thirds of ATH5-expressing cells
(Fig. 6C). This was confirmed
by ATH5 overexpression, which resulted in a threefold increase in promoter
activity, as measured by CAT assay, and raised the proportion of
lac+ cells to ∼30%, suggesting that upregulation of
promoter activity by ATH5 brings the whole population of ATH5-expressing cells
above the threshold for X-gal detection
(Fig. 6C). A similar analysis
was performed at stage 22-23 using NGN2 overexpression. Whereas at this stage∼
30% of retinal cells accumulate ATH5 transcripts, X-gal staining reveals
promoter activity in only ∼1% of cells
(Fig. 6B), the majority of
which are weakly stained (Fig.
6D). NGN2 overexpression enhanced ATH5 promoter activity about
10-fold to the level found at E6 (Fig.
6A), but only increased the proportion of cells stained with X-gal
to ∼6% (Fig. 6B,D). In most
ATH5-expressing cells, the level of promoter activity remains below the
threshold for detection by the X-gal reagent
(Fig. 6C).

A rapid decrease in the activity of the ATH5 promoter was detected between
E6 and E9, marking the transition between the second and third phases in the
regulation of ATH5 (Fig. 6A).
At stages 34 and 37, the promoter is poorly active and no longer responds to
overexpression of ATH5 or NGN2. Thus, it appears that the ability of the ATH5
protein efficiently to stimulate its own expression in a subset of competent
progenitors is restricted to the narrow time window (E5-E7) when most RGC
precursors are born.

Regulation of the ATH5 promoter during retinogenesis. (A) Retinal cells
isolated at stages 22 (E3) to 37 (E12) were transfected with an
ATH5-promoter/CAT-reporter plasmid singly or in combinations with ATH5 and/or
NGN2 expression vectors. Cells were assayed for CAT activity 24 hours after
transfection. ATH5 transcription is passing through three phases in the course
of retinogenesis. During the first phase (HH22-HH24), the promoter is weakly
active and responds strongly to NGN2 overexpression, except in the presence of
ATH5. During the second phase (HH25-HH30), upregulation of promoter activity
coincides with a transient increase in ATH5 mRNA (curve). ATH5 and NGN2 both
enhance promoter activity and ATH5 becomes dominant over NGN2. The third phase
(HH34 and beyond) sees a decrease in ATH5 mRNA and is marked by the inability
of either proneural protein to transactivate the promoter. (B) Retinal cells
isolated at stages 22-23 and 29-30 were transfected with an
ATH5-promoter/lacZ-reporter plasmid singly or in combinations with
NGN2 or ATH5 expression vectors. lac+ cells were counted
after 24 hours in culture. The number of lac+ cells
obtained upon transfection with a control SV40-promoter/lacZ-reporter
plasmid at each stage is set at 100 and cell numbers are given relative to
this value. (C) Schematic representation of promoter activity as revealed by
X-gal and CAT assays. Approximately 30% of cells express ATH5 at stages 22-23
and 29-30. The horizontal arrows indicate average promoter activity as
measured by CAT assay, the open arrowhead marks the threshold for X-gal
detection. At stage 22-23, promoter activity is low and only one in 30
ATH5-expressing cells is detected by X-gal. Overexpression of NGN2 increases
promoter activity 10-fold but only six out of 30 ATH5-expressing cells are
stained with X-gal. At stage 29-30, the whole population of ATH5-expressing
cells is stained with X-GAL upon ATH5 overexpression. (D) At stage 22-23, most
cells are weakly stained with X-GAL (arrowheads in a). Overexpression of NGN2
strongly enhances promoter activity (b) and the number of X-gal stained cells.
At stage 29-30, cells display strong promoter activity (c) and overexpression
of ATH5 enhances staining intensity (d).

HES1 is a dominant-negative regulator of the ATH5 promoter

Next, we asked why ATH5 does not efficiently stimulate its own expression
at early stages of retinogenesis. The mutually exclusive domains of ATH5 and
HES1 in early retina (Figs 1,
2) suggested that HES1
interfered negatively with ATH5 expression. Inhibition of the ATH5 promoter by
HES1 was demonstrated by co-transfection of an ATH5 promoter/CAT reporter
plasmid and a HES1 expression vector in retinal cells at stages 22-23 and 30.
At both stages, promoter activity was reduced to the background level
(Fig. 7A). We tested whether
HES1 overexpression influences activation by transfected NGN2 and ATH5. We
found both in electroporated retina and in transfected retinal cells that HES1
overexpression prevents activation of the ATH5 promoter by either proneural
proteins, indicating that HES1 acts as a dominant-negative effector
(Fig. 3F,
Fig. 7A). We then assessed
whether endogenously expressed HES1 is able to block the NGN2-mediated
activation of the ATH5 gene. We separated the central from the peripheral
regions of retinas at stage 22-23, electroporated the tissues with either a
NGN2 or an empty expression vector, and cultured them as explants for 24 hours
(Fig. 7B). As revealed by
northern blot hybridisation, overexpression of NGN2 led to accumulation of
endogenous ATH5 mRNA in the central but not in the peripheral retina,
indicating that NGN2 is able to stimulate ATH5 transcription where most cells
express HES1 at low levels, whereas the high rate of HES1 expression at the
periphery precludes activation of the ATH5 gene. Consistent with this view,
very few lac+ cells were found at the periphery of a
retina electroporated with a mix of ATH5-promoter/lac reporter
plasmid and NGN2 expression vector (Fig.
3E), whereas the number of lac+ cells was much
increased in the central region (Fig.
3D). To quantify the strength of the HES1 inhibition, stage 22-23
retinal cells were co-transfected with different ratios of the NGN2 and HES1
expression vectors. We found that above a ratio of 2:1, NGN2 overcomes the
inhibitory effect of HES1 and activates the ATH5 promoter
(Fig. 7A). We then reasoned
that if the inhibitory interactions between HES1 and the proneural proteins
are concentration dependent, they could account for the expression pattern at
the domain boundary where, from stage 23 onwards, HES1 is downregulated and
NGN2 expression expands ahead of the ATH5 domain
(Fig. 2H-K). To test this
prediction, stage 24 retinal cells were transfected with the
ATH5-promoter/lacZ reporter, either alone or together with an ATH5 or
NGN2 expression vector. Twenty-four hours later, lac+
cells were revealed and cells were processed for in situ hybridisation with a
35S-labelled HES1-specific riboprobe. Overexpression of NGN2
increased the relative number of double-labelled cells, whereas ATH5 did not
(Fig. 7C). Congruent with the
in vivo expression pattern, NGN2 overexpression was able to activate the ATH5
promoter in cells expressing HES1 at low levels, whereas ATH5 could not
(Fig. 7C). These results
suggest that NGN2, being less sensitive than ATH5 to HES1 inhibition,
contributes to the expansion of the ATH5 domain when expression of HES1 begins
to wane. Moreover, this finding implies that ATH5 expression is driven by
NGN2, rather than by ATH5, in cells co-expressing HES1 and ATH5
(Fig. 4). Finally, the
inability of ATH5 to activate its own promoter in the presence of HES1
explains why ATH5 is a weak activator at early stages: the low but significant
expression of HES1 in the central retina between stages 22 and 25 is probably
sufficient to prevent autostimulation.

HES1 exerts a dominant-negative effect upon the ATH5 promoter. (A) Retinal
cells at stages 22-23 or 29-30 were transfected with an
ATH5-promoter/CAT-reporter plasmid alone or with different combinations of the
ATH5, NGN2 and HES1 expression vectors. NGN2 and HES1 expression vectors were
co-transfected in different ratios, as indicated. (B) Peripheral and central
regions of retina were dissected at stage 22-23. They were electroporated with
NGN2 and control expression vectors and cultured as explants for 24 hours. The
presence of ATH5 mRNA was detected by northern blot hybridisation.
Overexpression of NGN2 upregulated ATH5 expression in the central but not in
the peripheral retina. (C) Retinal cells at stages 24 or 29-30 were
transfected with an ATH5-promoter/lacZ-reporter plasmid singly or in
combinations with ATH5 or NGN2 expression vectors. lac+
cells were revealed and processed for in situ hybridization with a
HES1-specific riboprobe. Overexpression of NGN2 increased the relative number
of double-labelled cells, indicating that the NGN2 protein can activate the
ATH5 promoter in cells that express HES1 (a), unlike the ATH5 protein (b).

ATH5 and NGN2 compete to regulate the ATH5 promoter

In early retina, NGN2 is a strong activator of ATH5 transcription, whereas
overexpression of ATH5 only results in a modest increase in promoter activity.
Conversely, ATH5 is a more potent activator than NGN2 at stage 29-30
(Fig. 6A). We therefore
wondered what effects would the overexpression of both transcription factors
have on the activity of the ATH5 promoter. When retinal cells were
co-transfected at stage 22-23 with an ATH5-promoter/CAT-reporter plasmid and
expression vectors encoding both the NGN2 and ATH5 proteins, stimulation by
NGN2 was abolished and promoter activity remained low. By contrast,
overexpression of ATH5 at stage 30 demonstrates that ATH5 has now become an
efficient positive regulator of its own promoter: at this stage, the promoter
is upregulated to the same high level whether ATH5 is overexpressed alone or
in combination with NGN2 (Fig.
6A).

These results suggest that ATH5 and NGN2 may compete for the same
regulatory elements, and that ATH5 is the dominant activator in the absence of
HES1. At early stages, because of the presence of HES1, ATH5 cannot upregulate
its own expression but can efficiently compete with NGN2. Mutational analysis
showing that the two proteins are using the same E-box elements to mediate
their effects (Fig. 1S) and
chromatin immunoprecipitation (ChIP) experiments demonstrating that both the
ATH5 and NGN2 proteins bind the ATH5 promoter in vivo at stage 22-23 and stage
29-30 (Skowronska-Krawczyk et al.,
2004) are in favour of this hypothesis. In addition, the
co-transfection results suggest that autostimulation is in large part
responsible for the upregulation of ATH5 expression at stage 30, whereas at
earlier stages ATH5 may contribute to the downregulation of its own
expression.

The presence of ATH5 transcripts in ∼26% of stage 18 neuroepithelial
cells (Fig. 1)
(Skowronska-Krawczyk et al.,
2004) and of HES1 transcripts in most of the ATH5-expressing cells
at stage 22-23 (Fig. 4), taken
together with previous findings (Liu et
al., 2001; Matter-Sadzinski et
al., 2001), provide ample evidence that the endogenous
ATH5 gene is transcribed in proliferating cells. The differential
response of the ATH5 promoter to the NGN2 and ATH5 proteins in the course of
retina development (Fig. 6) and
the different sensitivities of these proteins towards HES1
(Fig. 7) suggest their
implication at distinct moments in progenitor commitment to the RGC fate. To
analyse how stimulation of the ATH5 promoter correlates with the proliferative
status, NGN2 or ATH5 expression vectors were transfected together with an ATH5
promoter/lacZ-reporter plasmid at the stages when each factor exerts
its major effect and the transfected cells were pulsed with
[3H]-thymidine at the end of a 24-hour culture period
(Fig. 8A). Forced expression of
NGN2 at stage 22-23 led to a 3.5-fold increase in double-labelled cells and to
a sevenfold enlargement of the non-radioactive lac+ cell
population. On the whole, NGN2 overexpression diminished by half the
[3H]-thymidine-labelling index of cells that had an active ATH5
promoter when compared with controls (i.e. from ∼34% to ∼17%). This
result reveals the dual effect of overexpressed NGN2, which both stimulates
ATH5 expression in proliferating cells and significantly increases the pool of
non-dividing cells that have an upregulated ATH5 promoter
(Fig. 8A), indicating that NGN2
drives cells out of the S phase.

ATH5 expression is upregulated during the last S phase. (A) Retinal cells
isolated at stages 22-23 and 28-29 were transfected with an
ATH5-promoter/lacZ-reporter plasmid singly or in combinations with
NGN2 or ATH5 expression vectors and pulse-labelled with
[3H]-thymidine at the end of a 24-hour culture period. (Left) The
number of lac+ cells counted when the reporter plasmid was
transfected alone is set at 1. (Right) At stage 22-23, overexpression of NGN2
enhances promoter activity in proliferating cells (a) and increases the pool
of nonradioactive cells whose ATH5 promoter is upregulated (b). At stage
28-29, lac+ cells whose promoter is strongly upregulated
are unlabelled (d). The detection of double-labelled cells (c) and their
increased number upon ATH5 overexpression indicate that ATH5 promoter activity
is upregulated during the S phase. (B, left) A retina at stage 29-30 was
pulse-labelled for 45 minutes with BrdU and chased for 15 minutes. Transverse
sections were hybridized with an ATH5-specific riboprobe. Most BrdU-positive
cells are in S phase and their nuclei are located on the vitreous side (vi) of
the pz. ATH5 transcripts accumulate on the ventricular side (ve) of the pz in
the region where cells are in the G1 and G2 phases of the cell cycle. A few
BrdU-positive nuclei are located in this region (arrowheads). (Right)
Schematic of mitosis in the pz. Scale bar: 40 μm.

Single-cell transcription analysis has revealed a lag period between the
downregulation of HES1 and the upregulation of Neuro M, suggesting that the
ATH5 autostimulatory pathway might be activated before the onset of Neuro M
expression. In this context, we wanted to know at which point of the cell
cycle is autostimulation able to drive the upregulation of ATH5 expression.
When stage 28-29 retinal cells transfected with the ATH5 reporter plasmid were
pulsed with [3H]-thymidine for 1 hour, ∼25% of
lac+ cells were in S phase and forced expression of ATH5
led to a twofold increase of this fraction and to a 3.5-fold enlargement of
the population of non-radioactive cells
(Fig. 8A). Most cells (>90%)
that had robust promoter activity, as evidenced by strong X-gal staining, were
found in the pool of non-radioactive cells both in the control and after ATH5
overexpression, indicating that cells bearing an upregulated ATH5 promoter do
not re-enter the S phase (Fig.
8A). In situ hybridisation combined with anti-BrdU
immunohistochemistry revealed a strong accumulation of ATH5 transcripts within
the narrow region of the proliferative zone
(Fig. 2G,
Fig. 8B) where nuclei reside
during the G1- or G2-phases of the cell cycle. When a 45-minute BrdU pulse is
followed by a 15 minutes chase, the few BrdU-positive nuclei labelled with
silver grains have already moved back towards the ventricular side of the
retina (Fig. 8B, arrowheads).
Taken together, our results suggest that increased promoter activity via the
autostimulatory pathway begins during the last S phase and that the
accumulation of ATH5 transcripts peaks as cells leave the S phase and enter
the G2 phase.

Discussion

bHLH factors are required for the generation of a full array of the retinal
neurons, but how they contribute to neuroepithelium patterning, cell
commitment and cell cycle exit has remained elusive. Here, we use an approach
combining single-cell mRNA profiling and promoter analysis to clarify the
sequence of molecular events leading to neural progenitor commitment. In
particular, we define the transcriptional program determining the stages
through which neural cells progress as they convert from progenitors into
newborn RGCs. We find that spatial cell patterning and RGC commitment
correlate with the two main phases of ATH5 expression. During the period of
patterning, crossregulatory interactions between HES1, NGN2 and ATH5 keep ATH5
expression low, thereby maintaining the uncommitted status of ATH5-expressing
cells and enabling the expansion and intermingling of pools of progenitors
initially partitioned in distinct domains. Once progenitors are properly
distributed throughout the retina, about one-third of ATH5-expressing cells
become committed to acquire a definitive RGC fate immediately before exiting
the cell cycle. This requires a tight coordination between downregulation of
HES1, upregulation of NGN2, cell progression through the last S-phase and the
upregulation of ATH5. Cells that upregulate ATH5 expression initiate
transcription of early RGC-specific traits, then exit the cell cycle and
express Neuro M and other post-mitotic RGC-specific genes. Our study
highlights how changes in the transcriptional patterns correlate with the
progression of progenitors through the last cell cycle and with their
commitment to the RGC fate, underlining the role of HES1 as a key prompt of
the molecular events leading to RGC genesis.

Spatiotemporal progenitor patterning and RGC genesis

A specific feature of retinogenesis is that it proceeds from the centre to
the periphery such that all seven retinal cell types are distributed at the
proper ratio throughout the retina. At early stages of development, the
retinal neuroepithelium is subdivided into two developmentally distinct
territories. Low levels of HES1 transcripts outline a broad region of the
posterior retina where ATH5, NGN2 and ASH1 are expressed, whereas a robust
accumulation of HES1 transcripts throughout the anterior retina prevents the
onset of proneural gene expression. HES1 functions similarly at the onset of
neurogenesis in the olfactory placode, where it circumscribes a domain of
Mash1 expression (Cau et al.,
2000). It thus appears that HES1 is acting, much like
hairy in Drosophila, as a prepattern gene
(Skeath and Carroll, 1991).
Neurogenesis starts within a rather broad central region defined by expression
of ATH5, NGN2 and Neuro M. Cells expressing ATH5 at a high level and Neuro
M-positive cells are evenly distributed throughout the neurogenic domain,
indicating that the first newborn RGCs are produced with similar frequency
throughout the central retina. In the posterior retina, cells that initiated
expression of proneural genes are initially organized in two separate domains
corresponding to two retinal lineages: cells that express NGN2/ATH5 constitute
the progenitor pools from which early-born retinal neurons will emerge,
whereas ASH1-expressing cells form a pool for late-born neurons
(Brown et al., 1998;
Jasoni et al., 1994;
Matter-Sadzinski et al.,
2001). The opposite effects of NGN2 on ATH5 and ASH1 expression
combined with the inhibitory activity of ASH1 on ATH5 transcription
(Akagi et al., 2004;
Fode et al., 2000;
Matter-Sadzinski et al., 2001)
account for the distribution of ASH1 and ATH5/NGN2 cells in two distinct
progenitor domains, the more peripheral expression of ASH1 perhaps reflecting
its lower sensitivity towards HES1. The initial patterning of the posterior
retina resembles the neuroepithelial partitioning detected in other areas of
the developing CNS (Bertrand et al.,
2002; Jessell,
2000). However, whereas in other CNS regions the refining of
borders is essential for the precise spatial generation of different classes
of neurons along the dorsoventral axis, the blurring of borders and
intermingling of initially distinct progenitor pools are necessary for a
proper spatial distribution of neurons and glia throughout the retina.
Although ATH5/NGN2 and ASH1 expressions are mutually exclusive, a small
fraction of ATH5-expressing cells co-express ASH1
(Fig. 4)
(Matter-Sadzinski et al.,
2001), indicating that they are in a transient state prior to
acquiring a definite progenitor status. Because the ATH5, NGN2 and ASH1 genes
crossregulate and display different sensitivities towards HES1, we suppose
that various balances between these four factors may mediate alternate fate
choices. Such dynamic regulatory interactions are, in part, responsible for
the progressive loss of patterning in the posterior retina. The ATH5/NGN2
domain remains restricted to the posterior retina until E4 and expands to keep
pace with growth of the whole retina at a rate similar to that reported for
the differentiation of RGCs (McCabe et
al., 1999). Despite significant changes in the expression pattern
of ATH5, similar proportions of retinal cells express this gene at stages 18
and 29-30, suggesting that ATH5-expressing cells propagate at a rate
comparable with that of the other progenitors during the period of
patterning.

Even though the population of ATH5-expressing cells is established at E2.5,
only a small fraction of these will differentiate into RGCs until E4
(Prada et al., 1991;
Rager, 1980;
Waid and McLoon, 1995).
Retinogenesis is controlled by components of the Notch pathway
(Perron and Harris, 2000;
Vetter and Brown, 2001), which
may employ two strategies to keep the majority of cells in the central retina
from differentiating during the patterning period. Cells that express
proneural genes may promote the upregulation of HES1 in neighbouring cells,
thereby preventing them from expressing proneural genes. The proximity in
central retina of individual cells that highly express HES1 or ATH5 is indeed
indicative of ongoing lateral inhibition. However, cells strongly expressing
Notch effectors are rare in the posterior retina
(Fig. 1I,
Fig. 2L), whereas a high
proportion of ATH5-expressing progenitors co-express HES1
(Fig. 4). Thus, it appears that
the low level of HES1 in cells that have already initiated NGN2 and ATH5
expression suffices to prevent the upregulation of these genes. The
proliferative state is thereby maintained in most ATH5-expressing cells, as
required to ensure the proper ratio of RGC progenitors in the posterior retina
and as expected of HES genes, which function to keep neuroepithelial cells
undifferentiated, thereby regulating the size and cell architecture of brain
structures and retina (Hatakeyama et al.,
2004; Ishibashi et al.,
1995; Takatsuka et al.,
2004; Tomita et al.,
1996). In anterior retina, progenitor cell patterning becomes
evident by E4 and the expansion of proneural gene expression proceeds, much as
in zebrafish (Masai et al.,
2000), in a wave-like fashion as HES1 expression recedes to the
retinal margin. The ASH1 and NGN2 expression domains expand to the periphery
at similar rates, whereas the progression of the ATH5 domain is slightly
delayed (Fig. 2). The full
patterning of the retina accomplished around E6 coincides with the
upregulation of proneural gene expression throughout the retina and with the
peak of RGC production.

To analyse how ATH5 is regulated along the course of RGC specification, we
used a promoter region extending 775 bp upstream of the initiation codon. The
cloned sequence accurately reproduces the activity and the mode of regulation
of the endogenous promoter. It contains essential regulatory elements that are
well conserved across distant vertebrate species
(Brown et al., 2002;
Hutcheson et al., 2005;
Skowronska-Krawczyk et al.,
2004), but it is unclear whether the different species use similar
strategies to regulate ATH5 expression. Whereas a proximal cis-regulatory
region of the Xenopus Xath5 gene suffices, much as in the chick
retina, to drive retina specific reporter gene expression in a bHLH-dependent
manner, the mouse ATH5 promoter appears to be regulated differently
(Hutcheson et al., 2005). It
is tempting to speculate that the different modes regulating ATH5 across
species may account for differences in the spatiotemporal progenitor
patterning of the retinal neuroepithelium. Differences in the developments of
the anterior and posterior retinas may have permitted the evolution of a
specialized structure such as the macula.

Multiple functions of NGN2 in the specification of RGCs

Our study reveals that NGN2 acts at different regulatory levels during RGC
specification. In early retina, NGN2 is a principal regulator of ATH5
expression and exerts this function through direct activation of ATH5
transcription and through crossregulatory interactions with HES1. In addition,
NGN2 drives ATH5-expressing cells out of S phase. Whereas the capacity of NGN2
to promote cell cycle arrest is part of its panneuronal activities and is in
evidence in other compartments of the developing CNS
(Farah et al., 2000;
Ma et al., 1996;
Novitch et al., 2001), its
capacity to activate ATH5 expression is largely retina specific. The
quasi-simultaneous onset of NGN2 and ATH5 expression in the central retina
shortly after formation of the eye cup
(Fig. 1), the capacity of NGN2
to activate ATH5 transcription (Figs
6,
7)
(Matter-Sadzinski et al.,
2001) and to bind the ATH5 promoter
(Skowronska-Krawczyk et al.,
2004) at the early stages of development suggest that NGN2 may be
directly involved in the activation of ATH5 expression. Our finding that the
expansion of the NGN2 domain towards the anterior edge of the retina precedes
that of ATH5 argues in favour of this interpretation. In the retina of the
Ngn2–/– mouse, the much increased expression of ASH1
(Akagi et al., 2004) and the
downregulation of ATH5 (D. Skowronska-Krawczyk and J.M.M., unpublished) when
compared with the wild type, may result from an increase in the population of
ASH1-expressing cells at the expense of the ATH5/NGN2 progenitors, thus
underlining the importance of NGN2 in establishing and maintaining a pool of
ATH5-expressing cells. Both the NGN2 and ATH5 genes fail to be activated in
the retinal precursors of the Pax6–/– mouse and Pax6
has been proposed to regulate NGN2 directly in the mouse retina
(Brown et al., 1998;
Marquardt et al., 2001). There
are multiple E-boxes but no consensus Pax6 binding site in the chicken ATH5
promoter, and therefore we favour the idea that Pax6 regulates ATH5 via NGN2.
The expression of NGN2 in many regions of the nervous system anlage where ATH5
is not detected and the demonstration that recruitment of NGN2 on the ATH5
promoter is retina specific
(Skowronska-Krawczyk et al.,
2004) provide evidence that a retina-specific context accounts for
the capacity of NGN2 to activate ATH5 expression. The ability of bHLH factors
to regulate the development of distinct neurons has been proposed to depend
upon the cellular contexts in which they function
(Perron et al., 1999). In
retina, this context may be determined, among other possibilities, by the
balance between NGN2 and HES1, as we show that HES1 inhibits the NGN2-mediated
activation of ATH5 in a dose-dependent manner
(Fig. 7). Likewise, the
upregulation of NGN2 correlates with the dowregulation of HES1
(Fig. 2C)
(Matter-Sadzinski et al.,
2001). Moreover, single cell transcriptional analysis reveals that
overexpressing NGN2 diminishes the pool of cells that co-express ATH5 and HES1
(Fig. 4C), an indication that
NGN2 may contribute to the downregulation of HES1 in early neural progenitors,
thereby providing a cellular environment permissive for ATH5
autostimulation.

The upregulation of both NGN2 and ATH5 occurs later in development, around
E6, but by then ATH5 has become the main regulator of its own transcription
(Fig. 6)
(Matter-Sadzinski et al.,
2001). NGN2 occupies the ATH5 promoter similarly at E3 and at E6
(Skowronska-Krawczyk et al.,
2004), suggesting that it still directly participates in the
control of ATH5 transcription. However, its main contribution to ATH5
expression may occur through other, indirect regulatory pathways. As
ATH5-expressing progenitors exit the cell cycle, NGN2 promotes the expression
first of Neuro M and then of Neuro D
(Novitch et al., 2001;
Perron et al., 1999;
Roztocil et al., 1997) both
stimulators of ATH5 promoter activity
(Matter-Sadzinski et al.,
2001). These distinct functions of NGN2 in the ontogenesis of RGCs
illustrate how, depending on specific combinations of transcription factors
and of other cellular components, neurogenic proteins may contribute to
neuronal identity.

How does the retina prevent the differentiation of cells that have
initiated ATH5 expression and preserve an expanding pool of progenitors during
a period of highly dynamic patterning and considerable tissue growth? The
interplay between the molecular mechanisms underlying patterning and those
controlling the rate of RGC differentiation appears to be an integral part of
retina development. Among the different regulatory pathways that are involved,
the transcriptional network regulating ATH5 has a pivotal role. In early
retina, the ATH5 gene is transcribed at a low rate in ATH5-expressing
progenitors and its forced expression initiates the precocious transcription
of β3 in these cells
(Matter-Sadzinski et al.,
2001), indicating that they are competent for the expression of
this early RGC marker. The appropriate ATH5 dose is controlled through a
complex interplay between positive (NGN2) and negative (HES1) regulators. We
show that individual progenitors co-express HES1 and ATH5, and that HES1
represses the ATH5 promoter, thereby demonstrating that an effector of Notch
helps maintain a low rate of ATH5 transcription during progenitor patterning,
in agreement with the role of Xnotch pathway components in the regulation of
Xath5 function (Schneider et al.,
2001). The precise dose of HES1 is crucial during this period;
when the level of HES1 is too high expression of ATH5 is suppressed, whereas
the lack of HES1 leads to the precocious differentiation of retinal cells. In
the posterior retina, HES1 is expressed at levels that are high enough to
prevent ATH5 autostimulation, but low enough to allow NGN2-mediated expression
of ATH5 (Fig. 9). As revealed
by overexpression experiments, NGN2 is a potent activator of ATH5 in retinal
progenitors, but its low expression level normally leads to low ATH5 levels.
Moreover, NGN2 is counteracted by ATH5 itself, which acts as a
dominant-negative inhibitor of NGN2 in cells expressing HES1, thereby directly
contributing to the negative control of its own expression. This competition
between NGN2 and ATH5 may occur at the promoter level as both factors bind the
ATH5 promoter in stage 22-23 retinas
(Skowronska-Krawczyk et al.,
2004) and require the E-boxes E2 and E4 (see Fig. S1 in the
supplementary material). Although HES1 represses the ATH5 promoter and
prevents efficient autostimulation in retinal progenitors
(Fig. 7), it does not prevent
the binding of either NGN2 or ATH5 to the ATH5 promoter. We surmise that ATH5
activity at early stages may be repressed by heterodimerization with HES1, as
reported for other bHLH proteins (Alifragis
et al., 1997). This mechanism of inhibition is consistent with the
fact that overexpression of ATH5 in retinal progenitors does not overcome
HES1-mediated inhibition of the ATH5 promoter, whereas NGN2 does so in a
dose-dependent manner (Fig. 7).
Thus it appears that a subtle balance and interplay between NGN2, ATH5 and
HES1 is responsible for maintaining ATH5 expression below the level needed to
trigger cell commitment and direct expression of RGC markers.

Interacting transcriptional patterns as retinal cells go through three
consecutive phases during the conversion of progenitors into newborn RGCs.

RGC commitment requires the coordinated downregulation of HES1 and
upregulation of NGN2 and ATH5 during the last cell cycle

During early retina development, ∼30% of progenitors express ATH5 but
only a fraction of these become RGCs. When and how is this subset committed to
the RGC fate? On E6, at the peak of RGC genesis, ATH5-positive cells are
distributed throughout the whole retina and a robust accumulation of ATH5
transcripts takes place within the proliferative zone. This phase of
transcriptional regulation is characterized by a marked increase in ATH5
promoter activity as ATH5 autostimulation becomes dominant in the absence of
HES1. ATH5-expressing progenitors progressively downregulate HES1 between E5
and E6 (Fig. 4), yet only one
third of them will upregulate ATH5 on E6. We suppose that they are those that
overexpression experiments conducted on E5 reveal to be permissive for ATH5
autostimulation (Fig. 6A). They
may have been selected on E5 for RGC commitment because they were at the
appropriate phase of the cell cycle when downregulation of HES1 was initiated.
Whereas forced expression of NGN2 halves the
[3H]-thymidine-labelling index, it led to a fourfold decrease in
the pool of cells co-expressing ATH5 and HES1
(Fig. 7A,
Fig. 4C), suggesting that a
significant fraction of S-phase cells have completed the downregulation of
HES1. The accumulation of ATH5 transcripts in the proliferative zone where G2
and G1 cells reside and the presence of some S-phase cells expressing high
levels of ATH5 (Fig. 8)
indicate that ATH5 upregulation begins during the last S phase and peaks
before cell cycle exit. Taken together, our results suggest that
downregulation of HES1 and entry into S phase generate the proper conditions
for ATH5 autostimulation, causing the ATH5 protein to accumulate during a very
short period at levels sufficiently high to help RGC precursors withdraw from
the cell cycle (Fig. 8)
(Ohnuma et al., 2002) and
trigger or boost expression of RGC-specific target genes (e.g. BRN3C andβ
3, respectively; Fig. 9).
The expression of both β3 and BRN3C is stimulated when ATH5 is
overexpressed and their in vivo activation coincides with increased ATH5
transcription (Liu et al.,
2001; Matter-Sadzinski et al.,
2001). As a case in point, the in vivo binding of ATH5 on theβ
3 promoter and robust β3 promoter activity are detected when ATH5
is upregulated (Matter-Sadzinski et al.,
2001; Skowronska-Krawczyk et
al., 2004).

Our results position HES1 as an important prompt at distinct stages in the
sequence of events leading to the specification of RGCs. By interacting with
the pathways that regulate ATH5 transcription, it prevents high-level ATH5
expression at the time of progenitor patterning, a function essential for
establishing and maintaining the pool of ATH5-expressing cells. Its timely
downregulation during the last cell cycle releases the autostimulatory
activity of the ATH5 protein in a subset of progenitors and thus controls the
timing of their commitment and perhaps the size of their pool.

Cells that upregulate ATH5 exit the cell cycle
(Fig. 8) and start expressing
the post-mitotic factor Neuro M. There is a time lag between the
downregulation of HES1 and the expression of Neuro M
(Fig. 4). Overexpression of
NGN2 in early retina leads to the precocious accumulation of ATH5-positive
cells expressing neither HES1 nor Neuro M, and drives these progenitors out of
S phase, presumably in G2 or in G1, until they withdraw from the cell cycle
and start expressing Neuro M. Thus, although the onset of Neuro M expression
coincides with the transient upregulation of NGN2 and ATH5 in the developing
retina (Roztocil et al., 1997;
Matter-Sadzinski et al.,
2001), it appears not to be regulated directly by these proteins,
a notion supported by the absence of ATH5 and NGN2 binding on the Neuro M
promoter in developing retina
(Skowronska-Krawczyk et al.,
2004) (D. Skowronska-Krawczyk and J.M.M., unpublished) and by the
finding that induction of Xath3 transcription by Xngn1 requires de novo
protein synthesis (Perron et al.,
1999). This also suggests that additional factors are required for
Neuro M expression, which may not yet be present in early ATH5-expressing
cells. Overexpression of NGN2 prevents early ATH5 progenitors from re-entering
the S phase but is not sufficient to promote their precocious cell cycle exit.
Likewise, overexpression of Xath5 at early stages of Xenopus
retinogenesis produces extra RGCs that are all born at the appropriate time
(Ohnuma et al., 2002). Thus,
it is only when the coordinated upregulation of NGN2 and ATH5 coincide with
the build up of cdk inhibitors (Dyer and
Cepko, 2001; Ohnuma and
Harris, 2003) that progenitors may leave the cell cycle in G1,
enter G0 and begin expressing Neuro M. Expression of ATH5 remains strong in
newborn RGCs and at this stage Neuro M contributes to its regulation
(Fig. 9).

About two-thirds of ATH5-expressing cells fail to upregulate ATH5
expression and to acquire RGC traits. We reason that although these cells
suppressed HES1, they only accessed their last S phase on E6, too late for
properly upregulating ATH5 and thus missing the time-window when most RGCs are
produced. Cell-fate tracing experiments suggest that they may become other
retinal cell types (Yang et al.,
2003) and the finding that a fraction of progenitors co-express
ATH5 and ASH1 on E6 indicates ongoing alternate fate opportunities
(Fig. 4)
(Matter-Sadzinski et al.,
2001). Even though all uncommitted ATH5-expressing progenitors are
competent to upregulate ATH5 (Fig.
6C), not all of them can be made to adopt the RGC fate by forced
expression of ATH5 (Liu et al.,
2001). Overexpression of Xath5 at later stages of Xenopus
retinogenesis does not change the proportion of RGCs and increases the number
of photoreceptor and bipolar cells (Moore
et al., 2002). It has been proposed that the ability of bHLH
factors to promote the development of distinct retinal neurons depends upon
the timing of their expression and/or function
(Moore et al., 2002;
Morrow et al., 1999). If this
was the case, the set of genes regulated by ATH5 would change over time to
comprise genes specific for later-born neurons. Establishing the compendium of
ATH5 transcriptional targets should help answer the question of whether ATH5
is dedicated solely to the production of RGCs or whether it also promotes the
development of other retinal subtypes.

Acknowledgments

We thank Christine Alliod for expert technical assistance. We are grateful
to Dorota Skowronska-Krawczyk for sharing results before publication. The
Swiss National Science Foundation, the Marguerite Vuilleumier Foundation, the
ProVisu Foundation, the Jules Gonin Eye Hospital and the State of Geneva
support our laboratories. Monika Puzianowska-Kuznicka was the recipient of a
short term EMBO fellowship.

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